Phosphoproteomics-identified ERF110 affects ... - Plant Physiology

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Based on several seemingly conflicting results described above, it was speculated that
the Ser62-phosphorylated isoform of ERF110 would be required for bolting. To
examine this hypothesis, the Ser62 of ERF110 was substituted with either an amino
acid alanine (A) or an aspartic acid (D) using site-directed mutagenesis (see Materials
and Methods). The former and the latter point mutants are mimetic of the
de-phosphorylation and phosphorylation isoforms of ERF110, respectively. As Figure
6 reveals, in the presence of higher levels of ethylene (or ACC), the transgenic plants
over-expressing the wild-type ERF110 bolted within 23.4 ± 0.17 days, whereas
ERF110 S62A over-expressing plants had a delayed bolting (24.3 ± 0.36 days). In
contrast, ERF110 S62D over-expression transgenic mutants bolted by 22.9 ± 0.22 days
(Figure 6 and Table 1). These data suggest that Ser62-phosphorylation status is
involved in normal Arabidopsis bolting. Under conditions of exposure to higher levels
of ethylene that induce delayed bolting in Arabidopsis, the role of
Ser62-phopshorylated ERF110 becomes significant (Figure 6 and Table 1).
To determine how Ser62-phosphorylated ERF110 affects bolting, a quantitative
real-time reverse-transcription polymerase chain reaction (qRT-PCR) analysis was
performed to screen gene expression levels of 23 known flowering homeotic genes in
both the wild-type and ERF110 knockout lines, erf110-1 and erf110-2 (Supplementary
Figure 7). These homeotic genes are believed to act downstream of ERF110 in the
ethylene-delayed bolting process. Interestingly, four of these flowering homeotic
genes –– AP1 (APETALA1), VIN3 (VERNALIZATION INSENSITIVE 3), FT
(FLOWERING LOCUS T) and ELF8 (EARLY FLOWERING 8) –– were indeed found
to exhibit a significant change in gene expression levels and were selected as
downstream putative transcription activation targets for ERF110. The transcript of
AP1 –– a positive integrator of flowering signaling (Irish and Sussex, 1990) ––
showed a consistent association with the abundance of Ser62-phosphorylated ERF110
protein in Col-0 and the ctr1-1 and erf110 mutant lines (Figure 7). To confirm the
direct link between Ser62 phosphorylation and AP1 gene expression, ERF110 S62A and
ERF110 S62D transgenic mutants were also included in the investigation. Again, a
strong correlation was found between the Ser62-phosphorylated ERF110 isoform and
AP1 gene expression among the ACC-treated transgenic plants (Figure 7). The AP1